Thermo Scientific ILIC Separations Technical Guide ILIC Separations A Practical Guide to ILIC Mechanisms, Method Development and Troubleshooting
Table of Contents Introduction 3 Early istory 3 Typical ILIC Stationary Phases and Applications 4 ILIC Retention Mechanism 6 ILIC: Advantages 8 ILIC: Limitations 1 ILIC Method Development 11 Know Your Analyte for ILIC Method Development 12 Stationary Phase Selection 14 The Mobile Phase in ILIC 19 The Effect of Column Temperature on ILIC Retention 27 Start up Guide 29 Troubleshooting 31 Column Care in ILIC 33 Stationary Phase Characterization Details 34 References 43
Introduction ydrophilic Interaction Liquid Chromatography (ILIC) is the most successful approach for the retention and separation of polar compounds. ILIC has seen a substantial increase in popularity over the last decade, driven by the increase in the development of polar drugs within the pharmaceutical industry and also the growing field of metabolomics, which primarily involves the analysis of polar molecules. This technique has been described as reversed reversed phase [1], since in ILIC the stationary phase is polar and the aqueous portion of the mobile phase acts as the stronger solvent, which is the opposite of conventional reversed phase chromatography. There are many stationary phases that can be used in a ILIC mode and many phases are generically described as ILIC, independently of their chemistry. There is also a variety of misinformation regarding the use of this technique. So what column should be used? What are the best mobile phase starting conditions? What are the common issues in ILIC method development that need to be addressed? These are the type of questions that ILIC users face and will be addressed within this guide. A range of applications will also be described which will highlight the use of this technique and how it can be successfully implemented into a working laboratory. Early istory The ILIC mode of separation can be traced back to 1951, when Gregor et al. [2] described a waterenriched layer on an ion-exchange resin surface. The following year, Samuelson and Sjöström analyzed monosaccharides on an ion-exchange column [3]. Then, in 1954, Rückert and Samuelson [4] postulated that a stagnant water layer could be responsible for the uptake of analytes. Several years later, in 1975, the analysis of sugars was accomplished on amino columns [5, 6]. owadays, the existence of an semiimmobilized water-enriched layer on the polar stationary phase, combined with a partitioning equilibrium of analytes into the mobile phase are accepted as the dominant ILIC mechanisms [1, 7]. owever, it was not until 199 that the acronym ILIC was first introduced by Alpert [7]. emström and Irgum [1] have written a comprehensive review on the subject matter which supplies an excellent background to the technique and how it has developed. 3
Typical ILIC Stationary Phases and Applications In spite of its early beginnings, ILIC did not become widely recognized as a distinct chromatographic mode until it was 'rediscovered' by the scientific community in the early 2's [8]. The rising popularity of ILIC coincided with a wider availability of specifically designed ILIC stationary phases with diverse functionalities, which could offer different selectivity and higher retention for polar compounds [8]. owever, unmodified bare or hybrid silica materials are still the most popular phases. lica columns specifically intended for ILIC have been developed; these are packed and stored in aqueous/organic, which contrasts the more conventional use of silica in normal phase chromatography where the mobile phase would be a mixture of hexane and IPA solvents. lica materials have also become available in sub-2 µm fully porous particles, in superficially porous particles and as monolithic columns. Approximately 35% of the reported applications are being developed on bare silica, as illustrated below. ILIC phases vs % of applications 1% 4% Based on Scifinder Scholar 27 search of the Chemical Abstracts database 23 212 bare/hybrid silica zwitterionic amide diaol aminopropyl other cyanopropyl 35% 25% 9% 12% 14% From the data above, it can be seen that the second most employed type of ILIC material are the zwitterionic phases, a typical example of which is the sulfobetaine phase. The sulfobetaine zwitterion has both positive (quaternary ammonium) and negative (sulfonic acid) groups in a 1:1 ratio, so that the net surface charge is zero, as illustrated in the figure below: Sulfobetaine Structure C 3 + S - 3 3 C The sulfoalkylbetaine zwitterionic functionality was originally introduced into polymeric supports by Irgum et al. to prepare ion-exchange materials for the analysis of inorganic compounds and proteins [1]. Subsequently, a similar functionality was immobilized on silica substrates. It must be pointed out that, although the net surface charge is zero, the negative charge of the sulfonic acid at the distal end of the phase may introduce some degree of electrostatic interaction with charged analytes [9]. Irgum and his group have discussed a new type of zwitterionic phase, with phosphorylcholine groups grafted onto a polymeric substrate. This material has a positively charged ammonium group at its distal end [1]. Zwitterionic phases are successfully used for the analysis of charged and neutral species, regardless of the possible electrostatic interactions that can arise. 4
Amide-bonded silica phases are another popular choice in ILIC. The amide functionality, which is attached to the silica surface either through a propyl or proprietary linker, cannot be charged in the p range typical for ILIC mobile phases. Retention is therefore less affected by ion exchange interactions. Additionally, amide phases are less prone to irreversible adsorption (a problem often associated with aminopropyl phases) [11]. Amide columns also show good reproducibility and stability. ther popular ILIC phases include: 1. Diol material, which exhibit a considerable degree of hydrophilicity 2. Aminopropyl materials are used for ILIC separations, especially for carbohydrate analysis. They have, however, shown irreversible adsorption of analytes (at least for reducing sugars), due to the reactive nature of amino functionalities [11] 3. Cyano materials find limited applications in ILIC, as insufficient retention of most polar compounds is generally experienced. This is due to the fact that cyano groups do not have hydrogen bond donor capabilities and therefore are not very hydrophilic To monitor ILIC trends, Pontén et al. [12] conducted a literature survey for which the results are summarized below. It can be seen that the majority of the ILIC applications are found in the pharmaceutical and clinical markets with 46% of the research papers being published coming from these areas. Another contributing factor to the growing success of ILIC was its suitability for a large range of bioanalytical compounds that started to be considered at the time by the pharmaceutical industry because of their pharmacodynamic properties [1]. A significant number of publications (18%) relate to biotechnology applications, in particular to proteomics, metabonomics and glycomics. 25% of the applications are distributed between a variety of areas, with food analysis being a major contributor. ILIC application field distribution 5 45 4 35 ILIC use % 3 25 2 15 1 5 Pharma/clinical ther Bio Food Inorganic Application fields 5
ILIC Retention Mechanism ILIC can be described as a variation of Reversed Phase Liquid Chromatography (RPLC) performed using a polar stationary phase. The mobile phase employed in ILIC is highly organic in nature (>6% solvent, typically acetonitrile) containing a small percentage of aqueous solvent/buffer or other polar solvent. The water in the mobile phase forms an aqueous-rich layer adsorbed to the polar surface of the stationary phase. Y DR G I IC PARTI TI IL P + 3 + 3 2 + 3 AC - + 3 i - - - - EL E CT R TI AC E R YDR G E DIG B T S TAT I C I Polar analytes preferentially partition into this aqueous rich layer and evidence [13] suggests that they are retained through a complex, combination of: hydrophilic partitioning of the analyte between the aqueous-rich layer and the bulk of the mobile phase hydrogen bonding between polar functional groups and the stationary phase electrostatic interactions on ionized functional groups Additionally, under the appropriate experimental conditions van der Waals interactions between the hydrophobic portions of the bonded ligands of the stationary phase (or the siloxane groups, at very low organic solvent concentrations), and the non-polar part of the analytes can also be present. 6
The resulting elution order in ILIC is roughly the opposite of that observed in a reversed phase separation [14], as in the following chromatograms: Elution under ILIC conditions 1 ILIC P=25 bar 2 Column: Mobile Phase: Flow Rate: Inj. Volume: Accucore ILIC, 2.6 μm 15 4.6 mm 92:8 acetonitrile:1mm ammonium formate, p 3.2 3.5 ml/min 1 μl Temp.: 4 C Detection: Sample: UV at 23 nm 1. epinephrine 2. norepinephrine. 1 2 3 4 5 Time (min) Elution under RPLC conditions 2 1 RPLC P= 326 bar Column: Accucore aq, 2.6 μm 1 2.1 mm Mobile Phase: 5 mm ammonium formate, p 3.2 Flow Rate:.6 ml/min Temp.: 3 C Inj. Volume: Detection: 2 μl UV at 23 nm. 1 2 3 4 5 Time (min) In the examples given above the analytes were retained under both RPLC and ILIC modes. owever, when operating under RPLC conditions the analytes were only marginally retained and not fully resolved. When working in ILIC the analytes are baseline resolved with the added advantage of a considerably lower back pressure. 7
ILIC: Advantages The main benefits of ILIC can be summarized as follows: Retention of hydrophilic compounds that are difficult to retain in RPLC without the use of an ion pair agent Elution order is based primarily on analyte hydrophilicity, as opposed to hydrophobicity, so ILIC selectivity is complementary to RPLC selectivity. Mobile phases have high organic content which generally allows: Improved signal/noise ratio, (S/) with MS detection This is illustrated below. The use of ILIC is often highlighted as advantageous for use with ESI MS detection by virtue of the fact that the highly organic mobile phases ensure efficient desolvation, which in turn leads to lower detection limits. In the example shown below, in order to retain nicotine and cotinine using reversed phase chromatography, a highly aqueous mobile phase is required (in this case 98%). This results in low S/ ratio, even after adjustment of the electrospray conditions. When the same two compounds are analyzed using ILIC, the S/ ratio for nicotine is 15 times higher than in RPLC; the S/ ratio for cotinine is 5 times higher in ILIC than in RPLC, as illustrated in the following chromatograms. Extracted Mass Chromatograms for nicotine and cotinine in ILIC S/: 551 icotine S/: 8 Cotinine Column: Mobile phase: Detection: ypersil GLD ILIC, 5μm 15 2.1 mm 1:9 (v/v) ammonium formate 5 mm p 3.5 / acetonitrile +ESI. 1. 2. 3. 4. 5. Time (min). 1. 2. 3. 4. 5. Time (min) Extracted Mass Chromatograms for nicotine and cotinine in RPLC S/: 35 icotine Column: ypersil GLD, 5μm S/: 15 Cotinine 15 2.1 mm Mobile Phase: 98:2 (v/v) water / acetonitrile +.1% formic acid Detection: +ESI (spray conditions adjusted for higher aqueous content of mobile phase). 1. 2. 3. 4. 5. Time (min). 1. 2. 3. 4. 5. Time (min) 8
ther advantages that can be associated with this technique include: Low back pressures, due to the low viscosity of the organic-rich mobile phases. This is especially relevant when using sub-2 μm particles; lower back pressure allows for the use of high flow rates for fast analysis (a word of warning: the optimum performance may become compromised as it has been observed [13] that in ILIC higher efficiencies are achieved at lower flow rates than in RPLC. This phenomenon will be illustrated further on in the guide). Lower back pressure also means that the frictional heating effect which could be detrimental when using sub-2 µm particles in RPLC conditions is not an issue in ILIC, since the pressure is generally 2 3 times less than in RPLC [15]. Less peak tailing upon column overloading. This phenomenon is observed when peaks for very polar analytes (eluted under ILIC conditions) are compared to the peaks that can be obtained for these compounds on silica columns using highly aqueous conditions (in per aqueous liquid chromatography, PALC). Good retention factors can be achieved under PALC conditions (by taking advantage of the hydrophobic character of siloxane groups at the surface of the silica), however the underlying adsorption mechanism is heterogeneous and involves active adsorption sites; this results in column overloading being experienced when analyzing strongly retained samples (k > 2) [16]. Compatibility with SPE extracts, if in 1% aprotic organic solvent. These can be directly injected without the need to dry down and reconstitute in mobile phase. nce the organic solvents are weak eluents under ILIC conditions, polar analytes are accumulated on a narrow zone near the column inlet (phenomenon described as sample on-column focusing) [17]. 9
ILIC: Limitations ne of the most common challenges in ILIC is distortion of peak shape that arises from mismatch of sample solvent and mobile phase as shown below Effect of sample solvent on peak shape, under ILIC conditions 1 Injection solvent: 1 mm 4 Ac = 7174 2 3 4 1 Injection solvent: Mobile phase 2 = 53975 3 4 Column: Mobile Phase: Flow Rate: Inj. Volume: ypersil GLD ILIC, 5 μm 15 4.6 mm 1:9 (v/v) ammonium acetate 1 mm, p 5./acetonitrile.6 ml/min 1 μl Temp.: 3 C Detection: Sample: UV at 258 nm 1. Benzophenone 2. Uracil 3. Cytosine 4. Acyclovir Injection solvent: 1 m 4 Ac/AC (5:5) 1 = 1475 2 Injection solvent: AC = 53995 2 3 4 1 3 4 Using an aqueous sample solvent (with high elution strength), which impairs the partitioning of the analytes into the stationary phase is detrimental to the chromatographic peak shapes. ther negative side effects that could be experienced if using an aqueous sample solvent are easy column overload, reduced retention and resolution. To ensure good chromatographic performance it is recommended that the sample solvent has an organic content greater than 5% [17]. owever, this can cause solubility issues with highly polar analytes. Johnson et al. addressed this problem by using an injection loop partially filled with a central sample plug, surrounded by the focusing organic solvent [18]. ther challenges associated with ILIC are: ILIC mechanisms are kinetically slower than the common mechanisms in RPLC, resulting in longer column equilibration times (typically two to four times longer than in RPLC). Accurate measurements of mobile phase p cannot be made when using high proportions of organic solvent. McCalley [19] showed that acetonitrile/ammonium formate 85/15 (v/v), where the aqueous buffer p was 3, presented in fact a p of about 5.2 to the column and analytes.* There is a common misconception that all ILIC columns will retain polar compounds in a similar manner. Indeed, one of the most recurrent uncertainties in ILIC method development is the difficulty in identifying the most suitable column to use. 1 *Although mentioned here, this issue will not be discussed further in this guide. When reference to mobile phase p is made, we always refer to the p of the aqueous part.
ILIC Method Development A systematic approach to method development should be taken when developing a method in ILIC. Method parameters should be considered and adjusted sequentially, one at the time. This section provides some guidelines for: which type of stationary phase is the most appropriate to use what effects the mobile phase composition and p can have on the separation how the column temperature can influence ILIC analysis. The ILIC method development process can be summarized in the flow chart below (and on page 3), which outlines the necessary sequential method development steps in a logical manner. Determine analyte log D, pka, solubility For a review of Column Group 1 and Column Group 2 see page 17 Log D> 1 ILIC not applicable Preliminary Method Development Steps What is the analyte overall charge eutral, zwitterionic or mixture of acids + bases -ve charged (acidic) +ve charged (basic) Column Group 1: zwitterion, amide, urea or Group 2: anion exchanger, silica Column Group 1: zwitterion or amide or Group 2a: anion exchanger Column Group 1: zwitterion, amide, urea or Group 2b: silica Initial mobile phase: Isocratic AC/1 mm 4Ac 8/2 Buffer p: 4.8 Initial mobile phase: Isocratic AC/1 mm 4Ac 8/2 Buffer p: 6.4 Initial mobile phase: Isocratic AC/1 mm 4Ac 8/2 Buffer p: 3.3 o Does the analyte have suitable retention time Yes o Are analytes separated Increase AC content Change in 1-5% steps Increase AC content Change in 1-5% steps Yes Change p Use pk s and the molecular state required to determine what p (charged or uncharged) Change p Use pk 3 s and the molecular state required to determine what p (charged or uncharged) Working Assay Change buffer concentration/buffer type Typical ranges: 5-2 mm for mid-polar to polar compounds 1-2 mm for very polar compounds Change in 2.5 mm steps Change buffer concentration/buffer type Typical ranges: 5-2 mm for mid-polar to polar compounds 1-2 mm for very polar compounds Change in 2.5 mm steps Method ptimization Steps Change column type but within same group Change column type but within same group Change column temperature 11
Know your analyte for ILIC Method Development Knowledge of the compound structure and the physiochemical properties of the molecules under investigation will aid the choice of column, as it will determine the possible retention mechanisms. Fine-tuning of the separation by optimizing the organic solvent content, the buffer concentration and the mobile phase p should follow. Understanding the physiochemical properties of the analyte is therefore essential to the selection of a suitable ILIC column. Firstly, gather information on the hydrophobic character of the analyte, provided by its log P (or log D) value. The partition coefficient, log P, is used for neutral compounds, or where the compound exists in a single form. Log P can be calculated from the ratio of concentrations of an un-ionized analyte in the two phases of a mixture of immiscible solvents (octanol and water being the most commonly used): log P oct/water = log [Analyte] octanol [Analyte] water Log P indicates the degree of hydrophobicity of a compound. A log P> denotes a relatively hydrophobic compound; a log P< denotes a more hydrophilic compound. ILIC is generally recommended for hydrophilic compounds with negative log P values. Another important analyte parameter is the pk a (-log K a ); K a gives us an indication of the acidity of a molecule. K a is the equilibrium constant for a chemical reaction and in the context of acid-base reactions is known as dissociation constant*. The equilibrium can be written symbolically as: A A - + + Where A is a generic acid that dissociates by splitting into: A, the conjugate base of the acid. +, the hydrogen ion or proton, which, in the case of aqueous solutions, exists as the solvated proton. The chemical species A, A and + are said to be in equilibrium when their concentrations do not change with the passing of time. The dissociation constant is the ratio of the equilibrium concentrations (in mol/l), denoted by [A], [A ] and [ + ]: K a = [A] The negative logarithmic measure of K a is generally used, due to the many orders of magnitude spanned by K a values. 12
The larger the pk a value, the weaker the acid. For ionizable solutes, the compound may exist as several different species in each phase at any given p. The most appropriate descriptor for ionizable compounds is the distribution coefficient, log D, since it is a measure of the p-dependant differential solubility of all species in the octanol/water system. Log D is the ratio of the sum of the concentrations of all forms of the analyte in each of the two phases: [ionized Analyte] log D oct/water = log [ionized Analyte] octanol water + [un ionized Analyte] + [un ionized Analyte] water Log D provides a good indication of the ionization state of a compound in solution at a specific p and is more representative of the hydrophobic character in buffered conditions than log P. Knowledge of the combination of log P, log D and pk a data for the analytes will help to guide the stationary phase selection process. octanol *ote: k b, the equilibrium constant for a base gives us an indication of the basicity of a molecule. istorically, k b has been defined as the association constant for protonation of the base B, to form the conjugate acid, B +. B + 2 B + + - K b= [B + ] + [ - ] [B] pk b 14-pk a 13
Stationary Phase Selection nce the physiochemical properties of the analyte are known, it is then suggested to match the analyte log D values to the degree of polarity of the ILIC phases. In general terms, the more negative the log D value for an analyte, the greater the degree of stationary phase polarity required to retain it. The following chart, which illustrates the relative hydrophilicity and ion-exchange properties for Thermo Scientific ILIC columns, can be used as a guide in stationary phase selection at this stage: Increased retention of bases Syncronis silica ypersil GLD silica Basic analytes Accucore ILIC Increased retention of acids Acclaim ILIC-1 Accucore Urea-ILIC eutral or charged analytes ypersil GLD ILIC Syncronis ILIC Accucore 15 -Amide-ILIC 1.4 1.5 1.6 1.7 1.8 1.9 2. 2.1 2.2 2.3 Acidic analytes Increased retention of polar compounds (degree of hydrophilicity) Relative polarity and ion-exchange characteristics for various ILIC phases (based on study illustrated in next section) Stationary phase materials with a higher degree of hydrophilicity are shown on the right-hand side of the chart. So, for example Thermo Scientific Accucore 15-Amide-ILIC, being the most polar of these phases should be chosen for very polar analytes (which may not get enough retention on a lesser polar phase). Although in ILIC the predominant retention mechanism is partitioning, secondary electrostatic interactions can play an important role in the separation. With charged analytes it is possible to use the ion-exchange properties of the stationary phase to one s advantage. Materials with cation exchange properties are shown above the X-axis, whilst the phase with negative ion-exchange properties (an anion exchanger) is shown below. So, for example, Thermo Scientific Syncronis silica is a material with considerable cation exchange characteristics, and can be recommended for the analysis of basic compounds. owever, this material will not be suitable for the retention of acidic compounds*. n the other hand, Thermo Scientific ypersil GLD ILIC, which has anion exchange properties, can be considered for acids but will show reduced retention for basic compounds. The materials with no ion-exchange properties, Thermo Scientific Acclaim ILIC-1, Thermo Scientific Accucore Urea-ILIC, Syncronis ILIC and Accucore 15-Amide-ILIC are suitable for both charged and neutral analytes. These phases are therefore particularly useful where there may be a requirement to separate a mixture of acidic, basic and neutral species. Column selection is discussed in more detail in the following section. * ote: this might not be the case for the separation of a mixture of acids. 14
ILIC Phase Characterization: Understanding the Column Chemistry As we have seen in the previous section the stationary phases used in ILIC are quite diverse and users are often under the misconception that ILIC columns are interchangeable. A rigorous testing regime, such as that proposed by Tanaka et al. [2] is essential for the characterization and understanding of ILIC phases and should help to overcome this issue. Understanding the possible ILIC retention mechanisms involved for a particular column-solvent-analyte system is crucial in the process of selecting the right ILIC stationary phase leading to a successful application. ILIC stationary phases should be characterized for: degree of hydrophilicity, selectivity for hydrophilic-hydrophobic groups, selectivity for positional and conformational isomers, evaluation of electrostatic interactions, evaluation of the acidic-basic nature of the stationary phases. We performed such a study and the details of the testing conditions are described in the table on page 37. The data from this study show structure-selectivity relationships for the various ILIC phases and are discussed in the following sections. ILIC Selectivity Thermo Scientific columns for ILIC are available in both solid core and fully porous materials, with a variety of chemistries. The data sets generated from the characterization testing regime (as developed by Tanaka [2]) and mentioned above, highlighted important retention characteristics and differences. Stationary phase characteristics have been visually illustrated by radar graphs [2, 21], which allow to express multi-dimensional data in a two-dimensional format and ultimately allow to visually assess and compare columns. The separation factors obtained in the course of the assessment for the ILIC columns were therefore arranged in radar plots. Each axis of the radar plots represents one of the following parameters: 1. α (C2) degree of hydrophobicity 2. α () degree of hydrophilicity 3. α (V/A) separation factor for configurational isomers 4. α (2dG/3dG) separation factor for positional isomers 5. α (AX) degree of anion exchange interactions 6. α (CX) degree of cation exchange interactions 7. α (Tb/Tp) acidic-basic nature of the stationary phase 8. k U absolute retention ote that a selectivity value of 1 corresponds to no selectivity (since α = k 2 /k 1, α = 1 when k 1 = k 2 ). 15
The ion exchange properties have a major impact on the shape of the radar plots, and this allows for a separation of the ILIC stationary phases into two main groups: Group 1: For phases which have little ion-exchange activity. These materials can be used for neutral, zwitterionic and mixtures of acids and bases. Group 2: For phases which have considerable ion-exchange activity. Group 2 can be divided further in two subcategories: a) Anion exchange phases; recommended for the analysis of acids. b) Cation exchange phases; recommended for the analysis of bases. Group 1 Radar plots for ILIC stationary phases with no ion-exchange properties k U /2 k U /2 (Tb/Tp) (C2) (Tb/Tp) (C2) (CX) /2 () (CX) /2 () (AX) (2dG/3dG) (V/A) Syncronis ILIC (AX) (2dG/3dG) (V/A) Accucore 15-Amide-ILIC k U /2 k U /2 (Tb/Tp) (C2) (Tb/Tp) (C2) (CX) /2 () (CX) /2 () (AX) (V/A) (AX) (V/A) (2dG/3dG) Accucore Urea-ILIC (2dG/3dG) Acclaim ILIC-1 Group 2a Radar plot for ILIC stationary phase with anion exchange properties k U /2 (Tb/Tp) (C2) (CX) /2 () (AX) (V/A) (2dG/3dG) ypersil GLD ILIC 16
Group 2b Radar plots for ILIC stationary phases with high cation exchange properties k U /2 k U /2 (Tb/Tp) (C2) (Tb/Tp) (C2) (CX) /2 () (CX) /2 () (AX) (V/A) (AX) (V/A) (2dG/3dG) ypersil GLD lica (2dG/3dG) Accucore ILIC k U /2 (Tb/Tp) (C2) (CX) /2 () (AX) (V/A) (2dG/3dG) Syncronis lica Further information on the data derived from this characterization testing regime, and on the individual column chemistries is provided in the Column Characterization Detail section starting on page 35. Column Geometry As a general recommendation, for ILIC standard operations select a 1 or 15 mm long column, packed with 3 5 µm particles and with a 4.6 mm id, if using UV detection. If ILIC is coupled to MS or CAD detection, use a smaller column, e.g. 1 2.1 mm or 5 2.1 mm, packed with 3 5 µm particles (the reduction in column length should be undertaken after having considered sample complexity and detector selectivity). For higher separation efficiency and resolution we suggest either sub 2 µm fully porous particles or solid core materials or longer columns. The optimum flow rate is dependent on the column internal diameter and particle size. Some investigations carried out into the kinetic performance of 1 4.6 mm ILIC columns highlighted that for the same particle size and particle type the optimum flow rate also varies with the column chemistry. 17
The following van Deemter graphs where plate height,, is plotted versus linear flow velocity - report the results of this investigation. (μm) 12. Accucore ILIC Accucore 15-Amide-ILIC 11. 1. 9. 8. 7. 6. 5. 4. 3. 2...5 1. 1.5 2. 2.5 3. 3.5 4. Linear velocity (mm/s) Columns: Mobile Phase: Sample: ptimum Flow Rate: Accucore 15-Amide-ILIC Accucore ILIC 2.6 μm 1 4.6 mm 9/1 acetonitrile/1 mm ammonium formate (p 3.5) atenolol Accucore 15-Amide-ILIC,.7 1. ml/min Accucore ILIC,.9 1.7 ml/min With respect to the two solid core 2.6 μm particle size materials (Accucore15-Amide-ILIC and Accucore ILIC), it can be seen above that the bare silica has a flatter profile (lower value of the C term in the van Deemter equation) and hence a better preservation of high efficiency at high flow rates than the amide material. The optimum flow rate for the bare silica (Accucore ILIC) is in the range of.9 1.7 ml/min; however, even above 1.7 ml/min efficiency is not compromised as the increase in is minimal. The amide phase (Accucore15-Amide-ILIC) demonstrated an optimum flow rate range of.7 1. ml/min. Within its optimal flow rate range, the bare silica phase demonstrated slightly lower values (plate height) than the amide material. Lower values correspond to higher plate counts, making the bare silica material more efficient. The zwitterionic phase (5 μm particle size) investigated showed a prominent variation in efficiency with flow rate, as illustrated in the figure below: 26. 24. 22. 2. Syncronis ILIC Column: Mobile Phase: Sample: Syncronis ILIC, 5 μm 1 4.6 mm 9/1 acetonitrile/1 mm ammonium formate (p 3.5) atenolol (μm) 18. 16. 14. 12. ptimum Flow Rate: Syncronis ILIC,.6.8 ml/min 1...5 1. 1.5 2. 2.5 3. 3.5 Linear velocity (mm/s) The zwitterionic phase showed relatively high and mass transfer term values. The minimum plate height is similar to the values reported by McCalley [13] and emström and Irgum [1]. According to emström and Irgum, the relative thickness of the zwitterionic interactive layer could be the major contributor to the high mass transfer term (although the authors were referring to a polymeric rather than a monomeric coating). From the van Deemter plot reported above, it can be seen that the best performance on the zwitterionic phase is achieved at flow rates between.6.8 ml/min. 18
The Mobile Phase in ILIC rganic Content In ILIC the mobile phase is highly organic (generally 6-7%; at least 3% water is required). It has been demonstrated that besides the selection of a suitable column chemistry, the organic modifier/aqueous ratio is a major factor controlling the separation selectivity. An increase in the percentage of organic solvent leads to an increase in retention; this phenomenon is illustrated in the figures below. k uracil 1.6 1.4 1.2 1..8.6.4 Syncronis ILIC ypersil GLD ILIC ypersil GLD lica Accucore ILIC Syncronis lica Amide ILIC k uracil.9.7.5.3 ypercarb* Acclaim ILIC-1 Acclaim Mixed Mode ILIC-1.2. 1 2 3 4 5 6 7 8 9 1 % Acetonitrile (v/v).1 1 2 3 4 5 6 7 8 9 1 % Acetonitrile (v/v) *Although ypercarb is not a polar stationary phase, it was included in this study because of its dual behavior (RP and ILIC), depending on the percentage of organic in the mobile phase, which allows its use for the retention of polars in RP and ILIC. For most columns the test compound has little retention (mean k of.2) when the acetonitrile content is less than 6%. Acclaim ILIC-1, Acclaim Mixed Mode ILIC-1 and ypercarb, as seen in the plot on the right display both typical reversed phase and ILIC mode retention characteristics, depending on the percentage of organic solvent in the mobile phase. When acetonitrile concentrations are between 6 9%, uracil retention increases with the percentage of acetonitrile (ILIC mode of retention); between 5 3% acetonitrile, uracil retention decreases as the concentration of acetonitrile becomes greater (a reversed-phase retention behavior). Interestingly, Acclaim Mixed Mode ILIC-1 does not retain uracil when the percentage of acetonitrile is between 3 8. 19
rganic Solvent Type Although acetonitrile is the most popular solvent used in ILIC, several other polar, water-miscible organic modifiers can be used. The elutropic strength is generally the inverse of that observed in RPLC, with the relative solvent strength summarized in the following table. Solvent Chemical Formula Aprotic solvents Tetrahydrofuran (TF) / C 2 C 2 C 2 C 2 \ Increasing solvent elutropic strength in ILIC Acetone C 3 C(=) C 3 Acetonitrile (AC) C 3 C Protic solvents Iso-propanol (IPA) C 3 C ( ) C 3 Ethanol (Et) Methanol (Me) Water C 3 C 2 C 3 In ILIC, it is generally advised to use aprotic solvents (solvents which cannot exchange a proton). owever, it has been demonstrated that alcohols can be used as the weak eluent [22], although they will generally cause a decrease in the strength of the partitioning, leading to less retentivity; this is due to their hydrogen bonding interactions with the water molecules and hence the competition between the two to solvate the surface of the ILIC stationary phase [17, 23]. Periat et al. unequivocally proved that large amounts of alcohols caused drastic reduction in retention [15]. Methanol and IPA when used in mixtures with acetonitrile (8/2 AC/Me or AC/IPA) - are not able to provide significant changes in selectivity and lead to significant loss in kinetic performance and peak broadening [17]. In some instances where weakly polar analytes would precipitate in water-containing mobile phases, protic solvents replaced water (technique known as non-aqueous ILIC ) [24]. As outlined in the flow chart on page 11, the starting mobile phase we suggest consists of 8/2 acetonitrile/aqueous buffer; the elution strength is then adjusted until acceptable retention is achieved. Alternatively, a generic gradient can be run, starting from 95% acetonitrile, holding for 2 minutes or longer, according to column length - (to establish whether compounds are weakly retained in ILIC mode) and gradually increasing the aqueous buffer percentage to about 4% over 15 minutes (for a 1 mm long column). A two minute hold should follow, to establish whether compounds are strongly retained in ILIC. If using a gradient, it is vital to have a sufficiently long post gradient re-equilibration stage, to allow for the volume of the water layer (as advocated by the ILIC partitioning model) to re-establish its initial conditions. We generally recommend a post gradient re-equilibration of approximately 2 column volumes. 2
The following chromatogram illustrates how irreproducible chromatography, with drifting retention times is obtained when a post-gradient equilibration of 1 minute is carried out in between runs. Run 1 Run 2 1 minute post-gradient equilibration (equal to approximately.5 column volumes) Column: Mobile Phase A: Accucore 15-Amide-ILIC, 2.6 μm 1 2.1 mm acetonitrile Mobile Phase B: 1 mm ammonium formate (p 6.6) delivered in a 5 4% B gradient over 15 minutes at.1 ml/min, followed by 1 minute equilibration time 1 2 3 4 5 6 7 8 9 1 11 Time (min) n the other hand, reproducible chromatography is obtained when a suitable post-gradient equilibration is carried out in between runs. This is illustrated in the figure below, where a post-gradient equilibration time of 23.5 minutes (corresponding to 2 column volumes) leads to perfectly overlaid chromatograms. Run 1 Run 2 23.5 minutes post-gradient equilibration (equal to approximately 2 column volumes) Column: Mobile Phase A: Syncronis ILIC, 5 μm 1 2.1 mm acetonitrile Mobile Phase B: 1 mm ammonium formate (p 6.6) delivered in a 5 % B gradient over 15 minutes at.2 ml/min, followed by 23.5 minutes equilibration time..5 1. 1.5 2. 2.5 3. 3.5 4. 4.5 5. 5.5 6. 6.5 7. 7.5 8. 8.5 Time (min) Do I eed a Buffer? As a general guideline buffers are added to the mobile phase to reduce peak tailing and/or retention of charged analytes. Early research indicated that the absence of buffer salts resulted in excessively long retention times and very broad peaks on amide, cyclodextrin, cyano and amino-based columns [25]. Due to their good solubility in organic solvents, recommended buffers for ILIC are ammonium salts of acetic and formic acids. These buffers also have the advantage of being volatile for use with mass spectrometry and charged aerosol detection. ther salts with relatively high solubility in high organic levels are bicarbonate salts, triethylamine phosphate and sodium perchlorate, but the last two are not MS compatible. 21
Apart from these, most buffers used in reversed phase should be avoided in ILIC, due to their poor solubility in highly organic mobile phases*. Generally, negatively or positively charged stationary phases require higher concentrations of buffers than neutral or zwitterionic phases. Electrostatic interactions are secondary forces which can have important contributions to the retention in ILIC, since some polar compounds can be charged at the mobile phase p conditions typically used [8]. The presence of buffers in the mobile phase can reduce electrostatic interactions (both attractive and repulsive) between charged analytes and the stationary phase. When using a gradient, we recommend adding the buffer in both mobile phases, to allow constant buffer strength to be maintained throughout the run. For example, mobile phase A is made up of 1% aqueous buffer and mobile phase B should contain 95% acetonitrile and 5% aqueous buffer; both mobile phases could be prepared by adding 5% of concentrated aqueous buffer to either water or acetonitrile. We also suggest that the concentrated aqueous buffer should be a maximum of 1 mm but not above, to avoid this solution crashing out when added to acetonitrile. The use of a lower concentration buffer will also reduce any suppression effects that can be observed with electrospray sources either in CAD or in MS. * Please note that these are general recommendations. There are specific cases e.g. USP methods for sugars which suggest the use of phosphate buffers in 75% acetonitrile. Mobile Phase Buffer p In general, charged compounds are more hydrophilic, and therefore are more retained in ILIC. The plot below shows the retention factor of acetylsalicylic acid increasing with the buffer p, on bare silica and zwitterionic phases: The effect of mobile phase buffer p on the retention of acetylsalicylic acid k acetyl salicylic acid 3. 2.5 2 1.5 1.5 Accucore ILIC Syncronis ILIC ypersil GLD lica Columns: Accucore ILIC, 2.6 µm 1 4.6 mm Syncronis ILIC and ypersil GLD lica, 5 µm 1 4.6 mm Mobile Phase: 9/1 acetonitrile/1 mm ammonium formate (the mobile phase buffer p was measured before the addition of acetonitrile) Flow Rate: 1. ml/min Inj. Volume: 5 µl Temp.: 3 C Detection: 228 nm 2 3 4 5 6 7 Buffer p Acetylsalicylic acid has a pk a of 3.5; as the buffer p increases (p between 4 6.5), it becomes deprotonated and hence more retained. As the buffer p drops below 4, the proportion of protonated acetylsalicylic acid increases, leading to less retention*. * Interestingly there are two effects occurring with the retention of acetylsalicyclic acid, one is the increased polarity which gives an increase in retention and the second is the effect of the two charges of the stationary phase and also of the compound, which will have a slightly negative effect on the retention. 22
The charge state of the stationary phase can affect ILIC retention of ionizable compounds, depending on the mobile phase buffer p; electrostatic interactions between a charged stationary phase and a charged analyte can be critical, leading to drastic variations in retention. This concept was unequivocally demonstrated by Guo and Gaiki [8], when analyzing cytosine on an amino column. When the mobile phase buffer p approached the cytosine pk a1 value (~ 4.6), cytosine became positively charged, inducing electrostatic repulsion from the positively charged amino stationary phase and its retention dropped. The mobile phase buffer p can also affect the stationary phase charge state; this, for example is the case for bare silica phases, where the silanol ionization varies with the mobile phase buffer p. At p>4-5, the silanols are deprotonated, making the silica surface negatively charged. This will have an effect on the retention of positively charged analytes. This phenomenon is illustrated below with the increased retention for cytidine. The retention of cytidine increases as the buffer p increases from 3.3 to 6.4. As the mobile phase buffer p increases the proportion of deprotonated silanol groups also increases, leading to increasing electrostatic attractions between the positively charged cytidine and the increasingly negatively charged silica surface. It is therefore very important to fine tune the mobile phase buffer p, when using bare silica phases for the analysis of basic compounds, in order to avoid excessively long retention times and peak broadening. The effect of mobile phase buffer p on the retention of cytidine on a bare silica phase k cytidine 4.1 4 3.9 3.8 3.7 3.6 Column: ypersil GLD lica, 5 µm 1 4.6 mm Mobile Phase: Flow Rate: Inj. Volume: 5 µl Temp.: Detection: 9/1 acetonitrile/1 mm ammonium formate 1. ml/min 3 C 248 nm 3.5 3.4 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 Buffer p verlaid chromatograms relative to the separation of cytidine on a bare silica phase, showing an increased retention with the mobile phase p p 3.3 p 4. p 6.4 Column: ypersil GLD lica, 5 µm 1 4.6 mm Mobile Phase: Flow Rate: Inj. Volume: 5 µl Temp.: Detection: 9/1 acetonitrile/1 mm ammonium formate 1. ml/min 3 C 248 nm 2 2.2 2.4 2.6 2.8 3 3.2 3.4 3.6 Time (min) lica-based neutral phases (e.g. amide phases) can bear negative charges, due to residual silanol groups when working within a 3 6.5 p range [26]; this was demonstrated by the characterization testing carried out on the Accucore 15-Amide-ILIC, where some cation exchange capabilities were observed. See the column characterization details on page 34 for further information. 23
Mobile Phase Buffer Type and Concentration The effect of different types of ammonium salts on the retention of acid and basic model compounds was investigated on various ILIC chemistries [27]. Ammonium formate provided longer retention times for acidic compounds on an amino phase, due to the weaker eluting strength of the formate ion in the ion exchange interaction (as opposed to the acetate ion which has a greater neutralizing effect of the electrostatic attractions between the surface of the positively charged stationary phase and the negatively charged acid). Ammonium formate and ammonium acetate did not provide differences in retention of basic analytes on the amino column. o significant differences were observed between acetate and formate salt for neutral and zwitterionic phases. As already pointed out, buffer salts in ILIC mobile phases are very important in reducing electrostatic interactions between charged analytes and the stationary phase. When electrostatic interactions are prevalent, an increase in the salt concentration leads to a decrease in retention of charged solutes on the stationary phases of opposite charge. This phenomenon can be seen below, where the retention for salicylic acid (negatively charged) on ypersil GLD ILIC (positively charged) decreases as the buffer concentration increases. The effect of ammonium acetate concentration on the retention of salicylic acid on ypersil GLD ILIC (anion exchanger) k salicylic acid 1 9 8 7 6 5 4 3 2 1 2.5 5 7.5 1 12.5 15 17.5 2 Ammonium Acetate concentration (mm) Column: ypersil GLD ILIC, 5 µm 1 4.6 mm Mobile Phase: 9/1 acetonitrile/ammonium acetate Flow Rate: 1. ml/min Inj. Volume: 5 µl Temp.: 3 C Detection: 228 nm 24
This is also illustrated by the following chromatograms, which show the separation of an acidic mixture, with retention of the anionic analytes decreasing as the concentration of ammonium acetate increases. Separation of a mixture of acids on ypersil GLD ILIC (anion exchanger) 1 1 2 3 5 1 15 2 25 3 2 5 mm Ammonium acetate 1 mm Ammonium acetate Column: ypersil GLD ILIC, 5 µm 1 x 4.6 mm Mobile Phase: 9/1 acetonitrile/ammonium acetate Flow Rate: 1. ml/min Inj. Volume: 5 µl Temp.: 3 C Detection: 228 nm Sample: 1. Salicylamide 2. Salicylic acid 3. Aspirin 3 5 1 15 2 25 3 1 2 15 mm Ammonium acetate 3 5 1 15 2 25 3 1 2 2 mm Ammonium acetate 3 5 1 15 2 25 3 Time (min) From the chromatograms above it is also evident that the increased salt concentration is beneficial to the peak shape, with sharper peaks when 2 mm ammonium acetate is used. Increased salt concentrations result in increased retention of positively charged solutes on stationary phases with same charge, as demonstrated below, where the retention of cytosine and cytidine on an anion exchanger increases with the salt concentration. Enhanced hydrogen-bonding interactions (between the analyte and the stationary phase) are responsible for this behavior. The hydrogen-bonding interactions are facilitated by the increased population of solvated salt ions in the mobile phase (salting-out effect). The effect of ammonium acetate concentration on the retention of cytosine and cytidine on ypersil GLD ILIC (anion exchanger) k 7 6 5 4 3 k' Cytosine k' Cytidine Column: ypersil GLD ILIC, 5 µm 1 4.6 mm Mobile Phase: 9/1 acetonitrile/ammonium acetate Flow Rate: 1. ml/min Inj. Volume: 5 µl Temp.: 3 C Detection: 248 nm 2 1 2.5 5 7.5 1 12.5 15 17.5 2 Ammonium Acetate concentration (mm) 25
Separation of a mixture of bases on ypersil GLD ILIC (anion exchanger) 4 1 3 2 2.5 mm Ammonium acetate 2.5 5 7.5 1 12.5 15 17.5 2 1 mm Ammonium acetate Column: ypersil GLD ILIC, 5 µm 1 4.6 mm Mobile Phase: Flow Rate: Inj. Volume: 5 µl Temp.: Detection: Sample: 9/1 acetonitrile/ammonium acetate 1. ml/min 3 C 248 nm 1. Uracil 2. Uridine 3. Cytosine 4. Cytidine 2.5 5 7.5 1 12.5 15 17.5 2 15 mm Ammonium acetate 2.5 5 7.5 1 12.5 15 17.5 2 2 mm Ammonium acetate 2.5 5 7.5 1 12.5 15 17.5 2 Time (min) In summary, the recommendations we offered for mobile phase selection for ILIC applications are: Use acetonitrile or other polar, water-miscible organic modifiers. Remember that the elutropic strength is inverse to what observed in RPLC (as outlined in table on page 2). Aprotic solvents give longer retention than protic solvents. ave a high organic content, between 6 to 97%; a minimum of 3% water is necessary to ensure sufficient hydration of the stationary phase. An increase in organic solvent will lead to an increase in retention. Use buffer salts such as ammonium acetate and ammonium formate to avoid peak tailing and to control retention times of charged analytes. Buffer salts concentrations are 2 2 mm, although 2 mm is recommended for organic content of below 9% only. igher concentrations are not soluble in high levels of organic and could impair MS or CAD signals. When using gradients, buffer both mobile phases, do not run buffer gradients. Do not run gradients from 1% organic to 1% aqueous. The charge state of the stationary phase can affect ILIC retention of ionizable compounds, depending on the mobile phase p. 26
The Effect of Column Temperature on ILIC Retention Column temperature is an important parameter that can also affect retention of polar analytes in ILIC [27]. The equation that is often used is derived from chemical thermodynamics, where the equilibrium point is related to the temperature, and is referred to as the van t off equation. In a chromatography sense the relationship between column temperature and retention factor is often described by the following: ln k= - /RT + S /R + ln where: Δ = enthalpy of interaction between stationary/mobile phase and analyte ΔS = entropy of interaction between stationary/mobile phase and analyte R = universal gas constant T = column temperature in Kelvin n = phase ratio The van t off equation should also apply to ILIC if the retention mechanism is consistent, in which case the relationship between ln k and 1/T is linear [27]. Using the experimental plots of ln k versus 1/T, covering a temperature range between 2 7 C, we calculated the enthalpic and entropic contributions to the chromatographic retention of a series of test compounds (deriving - Δ from the slope and ΔS from the intercept respectively). Temperature was observed to affect the retention and separation in different fashions dependent on the column being tested. n the next page are van t off plots for salicylic acid and cytosine, on four different columns. It can be seen that a decrease in retention for salicylic acid indicating a negative heat of enthalpy and hence an exothermic reaction is observed as the column temperature is increased on the two bare silica materials: Accucore ILIC and ypersil GLD lica (although on Accucore ILIC the relationship is not linear, suggesting the presence of multiple retention mechanisms). n the zwitterionic phase Syncronis ILIC the retention of salicylic acid is more or less independent of temperature; this behavior suggests low enthalpic contribution and high entropic contribution to the retention. Interestingly, the retention increases with the temperature on ypersil GLD ILIC. Positive enthalpy was obtained for this anion exchanger, indicating an endothermic process of transferring salicylic acid from the mobile phase to the stationary phase. For ypersil GLD ILIC, the positive enthalpy of salicylic acid could also be the evidence of both ion exchange and partitioning processes taking place. 27
Van't off plot for salicylic acid 1. Syncronis ILIC ypersil GLD ILIC Accucore ILIC ypersil GLD lica Mobile Phase: 9/1 acetonitrile/1 mm ammonium acetate Flow Rate: 1. ml/min Inj. Volume: 5 µl Detection: 228 nm 1. ln k.36 Shown below is a decrease in retention for cytosine on the four stationary phases as the column temperature is increased. Linear van t off plots resulted from this study. egative retention enthalpy values were obtained, indicating an exothermic process of transferring cytosine from the mobile phase to the stationery phase. 1. Syncronis ILIC ypersil GLD ILIC Accucore ILIC ypersil GLD lica ln k.1.1..28.3.32.34 1/T (K) Van't off plot for cytosine Mobile Phase: 9/1 acetonitrile/1 mm ammonium acetate Flow Rate: 1. ml/min Inj. Volume: 5 µl Detection: 248 nm 1..28.3.32.34 1/T (K).36 Independently of its effect on the retention of test compounds, column temperature often positively affects the chromatographic efficiency due to lower viscosity and higher diffusivity. ur study demonstrated lower back pressures could be achieved when the temperature was raised, with average pressure dropping from 113 to 77 bar as the temperature was taken from 2 to 7 C. 28
Start Up Guide A systematic method development approach is provided in the main section of this guide and we suggest referring to it for a comprehensive strategy. As a general recommendation: Choose the column chemistry according to the nature of the interactions between analyte and stationary phase. For standard operations select a 1 or 15 mm long column, with a 4.6 mm id and packed with 3 5 µm particles. The column length can be changed if resolution needs optimizing. Smaller column diameter is recommended if ILIC is coupled to MS detection. ptimum flow rate varies from phase to phase and column id. Some investigations carried out into the kinetic performance of 1 x 4.6 mm ILIC columns highlighted that the bare silica material has a wider range of optimal flow rate conditions (.9-1.7 ml/min) than the amide or the zwitterionic materials (.7 1. and.6.8 ml/min, respectively). As outlined in the flow-chart reported on the next page, the starting mobile phase we suggest consists of 8/2 acetonitrile/aqueous buffer. Adjust the elution strength with small (<5%) changes in organic/aqueous ratio until acceptable retention is achieved. Alternatively, a generic gradient can be run (especially when separation of analytes with differing retention factors has to be achieved), starting from 95% acetonitrile, holding for 2 minutes (to establish whether compounds are weakly retained in ILIC mode) and gradually increasing the water percentage to about 4% over 15 minutes (for a 1 mm long column). A 2 minute hold should follow, to establish whether compounds are strongly retained in ILIC. We discourage to use fast gradients or gradients that run from 1% organic to 1% aqueous, as it would be very difficult to reach a dynamic equilibrium with reproducible retention times and besides, the latter would fall outside the ILIC realm. We would also suggest that the buffer concentration is maintained constant during the gradient (by adding the buffer to the organic portion as well as the aqueous), unless using charged stationary phases - where increasing the buffer concentration during the gradient would disrupt electrostatic interactions with the analyte. If using gradients, it is vital to have a sufficiently long post gradient re-equilibration stage, to allow for the volume of the water layer (as advocated by the ILIC partitioning model) to re-establish its initial conditions. It is recommended that a post gradient re-equilibration of approximately 2 column volumes is used. The same level of equilibration is also recommended for the initial column conditioning prior to the start of analysis. Failure to sufficiently equilibrate ILIC columns will result in irreproducible chromatography, with drifting retention times. 29
Determine analyte log D, pka, solubility Log D> 1 ILIC not applicable Preliminary Method Development Steps What is the analyte overall charge eutral, zwitterionic or mixture of acids + bases -ve charged (acidic) +ve charged (basic) Column Group 1: zwitterion, amide, urea or Group 2: anion exchanger, silica Column Group 1: zwitterion or amide or Group 2a: anion exchanger Column Group 1: zwitterion, amide, urea or Group 2b: silica Initial mobile phase: Isocratic AC/1 mm 4Ac 8/2 Buffer p: 4.8 Initial mobile phase: Isocratic AC/1 mm 4Ac 8/2 Buffer p: 6.4 Initial mobile phase: Isocratic AC/1 mm 4Ac 8/2 Buffer p: 3.3 o Does the analyte have suitable retention time Yes o Are analytes separated Increase AC content Change in 1-5% steps Increase AC content Change in 1-5% steps Yes Change p Use pk s and the molecular state required to determine what p (charged or uncharged) Change p Use pk 3 s and the molecular state required to determine what p (charged or uncharged) Working Assay Change buffer concentration/buffer type Typical ranges: 5-2 mm for mid-polar to polar compounds 1-2 mm for very polar compounds Change in 2.5 mm steps Change buffer concentration/buffer type Typical ranges: 5-2 mm for mid-polar to polar compounds 1-2 mm for very polar compounds Change in 2.5 mm steps Method ptimization Steps Change column type but within same group Change column type but within same group Change column temperature 3
Troubleshooting This section is intended as an aid in troubleshooting specific chromatographic problems that could occur during the running of ILIC separations. It is not meant to be an exhaustive guide to PLC column and instrument troubleshooting. Advice specific to your column will be found in the Product Manual. The following topics will be covered: Solubility Allowable buffers Retention reproducibility (peak retention drift) p extremes affecting column stability Injection solvent Injection volume Syringe wash Broad Peaks Possible Cause Injection solvent (sample solvent) too strong for the mobile phase Syringe/needle wash does not match mobile phase Injection volume too large Corrective Action To ensure good chromatographic performance, it is recommended for the sample solvent to be as close as possible to the initial mobile phase conditions, or at least to have an organic content greater than 5%. Using aqueous sample solvent, with high elution strength which impairs the partitioning of the analytes into the stationary phase is detrimental for the chromatography, resulting in peak broadening. This in turn could lead to column overload, reduced retention and loss in resolution. Unfortunately polar analytes often have low solubility in organic solvents, in this case it is recommended to substitute water with methanol. In extreme cases of solubility issues, even the aqueous portion of the mobile phase can be replaced by polar non-aqueous solvents, in which case the technique is referred to as non-aqueous ILIC chromatography. The solution used for washing the syringe and the injection needle should be matched to the mobile phase composition but without the buffers. Undesired band broadening will result if too much water is used in the wash. Pure organic solvents should be avoided as well, as they are not polar enough to remove the analytes. Injection of excessive sample volumes may cause column overloading, resulting in broad/tailing peaks or, in extreme cases flattened peaks. The recommend injection volumes are.5 5 µl, for a 2.1 mm ID column and 5 5 µl, for a 4.6 mm ID column. 31
Peak Retention Drift Possible Cause Column not fully equilibrated Column contamination Corrective Action ILIC columns are less tolerant of short equilibration times than RPLC columns, mainly because the water in the aqueous layer within the stationary phase derives from the mobile phase and takes time to establish itself. This is particularly true for gradient conditions; postgradient re-equilibrations of approximately 2 column volumes are recommended. Fast ILIC gradients and gradients that run from 1% organic to 1% aqueous are to be discouraged. ew columns need conditioning too and we recommend to run approximately 2 column volumes of mobile phase. A shift in peak retention or resolution, associated with a sudden increase in operating pressure beyond expected levels indicate column contamination. Reversing the flow direction of the column may be attempted to remove debris on the inlet frit (column effluent should go to waste, to avoid PLC system contamination). To remove strongly retained materials from the column, flush the column in the reverse direction with very strong (ILIC) solvents such as 5:5 methanol:water. Strong buffers may be needed to remove ionic contaminants. Allowable Buffers Possible Issues Solubility/MS compatibility We recommend the use of buffered eluents in ILIC, in order to minimize electrostatic interactions between charged analytes and deprotonated silanol groups of the stationary phase. A concentration range 5 2 mm is generally sufficient for most analytes. Ammonium acetate and ammonium formate are mostly recommended due to their high solubility in mobile phases with high organic content; their buffering range 3.8 5.8 for ammonium acetate and 2.7 4.7 for ammonium formate are suitable for most ILIC applications and additionally they are compatible with MS detection due to their volatility. If higher p is required, ammonium hydroxide and ammonium carbonate can be considered. Formic acid and acetic acid are valuable alternatives, due to their solubility even in very high concentrations of organic solvents. Phosphate buffers should not be used, as they are liable to precipitate, and tend to foul MS sources. 32
Column Care in ILIC As with any other column, to maximize ILIC column life ensure that samples and mobile phases are particle-free. We also recommend the use of guard columns or inline filters between the switching valve and the column. p Stability Washing Conditions Storing Conditions Thermo Scientific ILIC columns are silica based. A suitable p range is 2 8. Prior to storage, buffered solutions used during ILIC analysis should be washed out of the column (and the PLC system). A suitable wash solution is 6% organic solvent in water. ILIC columns should be stored under ILIC conditions. A mixture of organic solvent and water in a 9:1 ratio is recommended for storage. The Product Manual will give specific advice. Ensure the column end-fittings are sealed with end-plugs to prevent the stationary phase from drying. 33
Stationary Phase Characterization Details Details of characterization test solutes and their physiochemical properties (pk a and log D values obtained from www.chemspider.com). Chromatographic conditions as follows: mobile phase 9:1 (v/v) acetonitrile:ammonium acetate (2 mm on the column, p 4.7); flow rate:.5 ml/min; UV detection: 254 nm; Injection volume: 5 µl; column temperature: 3 C Chromatographic Probes Molecular Structure Variable pk a Log D Separation Factors C 3 Toulene t o marker na 2.72 all Uridine ydrophobic/ hydrophilic interaction 12.6-1.58 α(c 2 ), α(), k(u) 5-Methyluridine 3 C ydrophobic interaction 12. -1.2 α(c 2 ) 2'-Deoxyuridine ydrophilic interaction 13.9-1.26 α() 2 Adenosine Configurational isomers selectivity 13.9-1.3 α(v/a) Vidarabine Configurational isomers selectivity 13.9-1.2 α(v/a) 2 2'-Deoxyguanosine 2 Positional isomers selectivity 13.5-1.14 α(2dg/3dg) 3'-Deoxyguanosine 2 Positional isomers selectivity 13.5-1.14 α(2dg/3dg) Sodium p-toluenesulfonate,,trimethylphenylammonium chloride 3 C S a C C 3 Cl C 3 C 3 Anion exchange selectivity Cation exchange selectivity na.88 α(ax) na -2.31 α(cx) Uracil ydrophilic interaction 13.8-1.8 α(ax), α(cx) Theobromine C 3 C 3 Acidic-basic nature of stationary phase 1-1.6 α(tb/tp) 34 Theophylline 3 C C 3 Acidic-basic nature of stationary phase 8.6 -.5 α(tb/tp)
Syncronis ILIC k U /2 (Tb/Tp) (C2) 2 to 8 (CX) /2 () 1 (AX) (V/A) 5 (2dG/3dG) 1.7 µm, 3 µm, 5 µm 3 C C 3 S 3 The zwitterionic material offers the highest absolute retention and good hydrophobic, hydrophilic and isomeric selectivity. Low ion exchange properties are demonstrated by this neutral material. Syncronis ILIC provides enhanced retention of charged and neutral polar compounds. utstanding peak shape and sensitivity are demonstrated on this phase. Syncronis ILIC stationary phase carries a zwitterionic functional group of the sulfobetaine type, monomerically bonded to highly pure, high surface area silica. The sulfobetaine zwitterion has both positive (quaternary ammonium) and negative (sulfonic acid) groups in a 1:1 ratio, so that the net surface charge is zero. Due to counterbalanced electrostatic forces, which lead to weaker secondary electrostatic interactions with the analyte, lower buffer concentrations are required to disrupt these interactions. The charge density of this phase is p-independent and the optimization of the mobile phase p is solely dictated by the analyte. Relative Abundance 1 9 8 7 6 5 4 3 2. 1 9 8 7 6 5 4 3 2 1. 1 9 8 7 6 5 4 3 2 1. 1 9 8 7 6 5 4 3 2 1..5.5.5.5 Benzyldimethylphenylammonium chloride 1. 1.5 2. 2.5 eostigmine 1. 1.5 2. 2.5 Edrophonium 1. 1.5 2. 2.5 Pyridostigmine 1. 1.5 2. 2.5 Time (min) Analysis of edrophonium, neostigmine and pyridostigmine from plasma by LC-MS/MS Column: Syncronis ILIC, 1.7 µm, 1 mm 2.1 mm Part umber: 9752-1213 Mobile Phase: Gradient: Flow Rate: acetonitrile / ammonium formate, 1 mm, p 3.3 (9:1 v/v) 5 55% B in 6 min 5 µl/min Inj. Volume: 1 µl Temp.: 25 C Detection: MS 35
Accucore 15-Amide-ILIC ydrophobicity k U /2 Low p Range igh (Tb/Tp) (C2) Pore ze 2 to 8 14 (CX) /2 () 15 Å 3 Particle ze 2.6 µm (AX) (V/A) (2dG/3dG) C 2 C C= This material offers good hydrophobic, hydrophilic and isomeric selectivity and some cation exchange properties. 2 n Amide phase bonded onto 15 Å pore diameter solid core particles igh retention of a broad range of hydrophilic analytes in ILIC mode Recommended for hydrophilic biomolecules such as glycans The amide bonded phases provide strong hydrogen bonding interaction between the stationary phase and the analytes, resulting in unique selectivity compared to other ILIC phases. Combined with larger pore size of the solid core particles, Accucore 15-Amide-ILIC is well suited for separating a variety of hydrophilic molecules, including carbohydrates and peptides. As a result the Accucore 15-Amide-ILIC is an excellent choice for glycan separations. A slight amount of cation exchange properties are demonstrated by this slightly acidic phase, possibly due to the presence of residual deprotonated silanol groups (under the mobile phase p conditions used for the characterization testing). Counts 1 12 11 2 1 3 9 8 7 6 5 4 3 2 1-1 -2 A 4 5 3 35 4 45 Minutes 6 7 8 9 1 11 5 1 15 2 25 3 35 4 45 Time (min) 2-AB labelled dextran ladder 35 3 25 2 15 1 5 B 1 11 12 13 14 15 16 17 18 19 2 21 Column: Part umber: 16726-1213 Mobile Phase A: Accucore 15-Amide-ILIC, 2.6 μm, 1 2.1 mm Acetonitrile Mobile Phase B: 5 mm ammonium formate (p 4.5) Gradient: Time (min) B% 2 4 5 45 5 45.5 2 5 2 Flow Rate: Inj. Volume: 2 µl 5 µl 5 µl/min Temp.: 6 C Detection: Sample: Backpressure: Fluorescence excitation wavelength: 33 nm Fluorescence emission wavelength: 42 nm 2-aminobenzamide labelled dextranladder 11 bar (A) 2 μl injection of sample, where 11 glycans were separated. (B) 5 μl injection of sample, zoomed-in to the later part of the gradient rise. A further 1 glycans were detected. 36
Acclaim ILIC-1 k U /2 (Tb/Tp) (C2) 2 to 8 (CX) /2 () 3 µm, 5 µm 12 (AX) (V/A) (2dG/3dG) Functionality: Proprietary ydrophilic Layer This material offers good hydrophilic selectivity. Some amount of cation exchange properties are demonstrated by this slightly acidic phase. Acclaim ILIC-1 is based on high purity, fully porous silica covalently modified with a proprietary hydrophilic layer. The cation exchange properties identified on this material are due to the presence of residual deprotonated silanol groups (under the mobile phase p conditions used for the characterisation testing). Acclaim ILIC-1 provides good retention for charged and neutral polar compounds. This phase shows dual mode retention behavior, with a U-shaped retention profile as a function of acetonitrile content in the mobile phase. Increasing retention is demonstrated at both low and high levels of acetonitrile (RPLC and ILIC modes, respectively). mau 25 2 1 3 6 9 12 Time (min) Pharmaceuticals 2 3 4 5 Column: Part umber: 74257 Mobile Phase: Flow Rate: Inj. Volume: 2 µl Acclaim ILIC-1, 3 μm 15 mm 4.6 mm 9/1 v/v acetonitrile/1 mm (total) ammonium acetate, p 5 1 ml/min Temp.: 3 C Detection: UV at 23 nm Sample: 1. Acetaminophen.1 mg/ml 2. Salicylic acid.1 3. Aspirin.2 4. Penicillin G.1 5. Metformin.1 37
Accucore Urea-ILIC ydrophobicity k U /2 Low p Range igh (Tb/Tp) (C2) Pore ze 2 to 8 14 (CX) /2 () 8 Å 3 Particle ze 2.6 µm (AX) (V/A) (2dG/3dG) 2 This neutral material offers good hydrophilic selectivity and low ion exchange activity. The urea phase bonded onto solid core particles provides a unique selectivity for polar analytes. Accucore Urea-ILIC provides good retention for charged and neutral polar compounds. mau 6 2 4 1 3 2.2.4.6.8 1. 1.2 1.4 Time (min) Analgesic compounds Column: Accucore Urea-ILIC, 2.6 µm 1 2.1 mm Part umber: 27726-1213 Mobile Phase: Flow Rate: Inj. Volume: composition 1:8:1, A : B : C A: water B: acetonitrile C: 1 mm ammonium acetate adjusted to p 4.9 3 µl/min 2 µl into 1 µl partial loop mode Injection Wash Solvent: water:acetonitrile 2:8 Temp.: 35 C Detection: Backpressure: Run Time : UV at 23 nm 71 bar 2 minutes Acetaminophen Salicylic acid Aspirin t R A s t R A s R s t R A s R s Mean.76 1.474.98 1.33 2.359 1.1 1.318 3.264 CV %. 1.17.48.92.49..63.48 Data from eight replicate analyses of a mixture of acetaminophen, salicylic acid and aspirin Retention time (t R ), peak asymmetry (A s ), peak resolution (R s ) 38
ypersil GLD lica k U /2 (Tb/Tp) (C2) 2 to 8 (CX) /2 () 175 (AX) (V/A) (2dG/3dG) Functionality: Unbonded lica This bare silica material offers good hydrophilic selectivity and some isomeric selectivity. Very strong cation exchange properties are demonstrated by this acidic phase. ighly pure base deactivated silica media utstanding peak shape and sensitivity Besides being the underlying support for all ypersil GLD phases, ypersil GLD lica has wide applications under both ILIC and normal phase conditions. The cation exchange properties identified on this material are due to the presence of residual deprotonated silanol groups (under the mobile phase p conditions used for the characterization testing). ypersil GLD lica is recommended for the analysis of basic or neutral polar compounds. 39
Accucore ILIC ydrophobicity k U /2 Low p Range igh (Tb/Tp) (C2) 2 to 8 14 Pore ze (CX) /2 () 8Å 3 Particle ze 2.6 µm (AX) (V/A) (2dG/3dG) Functionality: Unbonded lica The cation exchange properties identified on this material are due to the presence of residual deprotonated silanol groups (under the mobile phase p conditions used for the characterization testing). Accucore ILIC is recommended for the analysis of basic or neutral polar compounds. This bare silica material offers good hydrophobic and hydrophilic selectivity. Good absolute retention and some isomeric selectivity are demonstrated. Very strong cation exchange properties exhibited by this acidic phase. mau 14 12 1 8 6 4 2 1 2 3 4 5 6 7.5 1 1.5 2 2.5 3 3.5 4 Time (min) Catecholamines Column: Accucore LIC 2.6 µm, 5 2.1 mm Part umber: 17526-5213 Mobile Phase: Flow Rate: Inj. Volume: 5 µl 85:15 (v/v) acetonitrile/1mm ammonium formate, p 3.2 2 ml/min Temp.: 4 C Detection: Backpressure: Analytes: UV at 28 nm 157 bar 1. Catechol 2. 5-IAA 3. DPAC 4. Serotonin 5. L-tyrosine 6. Dopamine 7. L-DPA 4
Syncronis lica k U /2 (Tb/Tp) (C2) 2 to 8 (CX) /2 () 1 1.7 µm, 3 µm, 5 µm (AX) (V/A) (2dG/3dG) Functionality: Unbonded lica This bare silica material offers good absolute retention, good hydrophobic and hydrophilic selectivity and some isomeric selectivity. Very strong cation exchange properties are demonstrated by this acidic phase. Syncronis lica offers highly pure, high surface area silica. Serves as a powerful and efficient tool for the chromatography of moderately polar acids or neutral compounds in both ILIC and normal phase mode. The cation exchange properties identified on this material are due to the presence of residual deprotonated silanol groups (under the mobile phase p conditions used for the characterization testing). 41
ypersil GLD ILIC k U /2 (Tb/Tp) (C2) 2 to 8 (CX) /2 () 175 (AX) (V/A) 6 (2dG/3dG) The tertiary amine functionality from the polyethyleneimine bonding of this phase shows anion exchange activity with good hydrophilic selectivity. n ypersil GLD ILIC is recommended for the analysis of acidic or neutral polar compounds. k salicylic acid 5 45 4 35 3 25 2 15 1 5-5 -1 2 1 1 2 3 4 5 6 7 8 9 1 11 12 13 14 15 16 17 18 19 Time (min) 2 Column: ypersil GLD ILIC, 5 µm 15 4.6 mm Part umber: 2655-15463 Mobile Phase: Flow Rate: 5 mm ammonium acetate buffer (p 5) / acetonitrile (1:9).4 ml/min Inj. Volume: 1 µl Temp.: 3 C Detection: Sample: 25 nm 1. Cyanuric acid 2. Melamine Melamine and cyanuric acid 42
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